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Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.

Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs or genes/proteins that stimulate or regulate the angiogenic process, can rely on an increasing number of experimental models. Among non-mammalian models, Zebrafish is adopted by an increasing number of research groups. Moreover, angiogenesis and vasculogenesis in invertebrates like the leech Hirudo medicinalis share a high degree of similarity with the same processes occurring in humans, both under the structural/functional and biochemical points of view. Interestingly, Hirudo angiogenic growth factor receptors respond to corresponding human/mammalian recombinant growth factors and cytokines; in addition, Hirudo endogenous angiogenic growth factors and receptors react with antibodies against their human/mammalian counterparts. Furthermore, as it will be shown in this review, Hirudo has the unique advantage of having a virtually avascular muscular body wall, whereas the reliability of such a peculiar feature as a model for physiologically vascularised mammalian tissues has to be thoroughly investigated. Hirudo has proven so far to allow unambiguous, clear-cut studies on the angiogenic potential of gene-products or drugs, as well as on the anti-angiogenic compounds. This article will review the biology of angiogenesis in Hirudo and the data so far collected on angiogenesis stimulation/modulation in this model; an example describing a study on the biological activity of a naked DNA vector for angiogenesis gene therapy will also be provided.

Vascular endothelial growth factor (VEGF) is fundamental in vertebrates for correct development of blood vessels. However, there are only few data about the presence of VEGF in invertebrates. In this study the role of VEGF in neovessel formation is investigated in Hirudo medicinalis. The leech is able to respond to administration of human VEGF by formation of new vessels. The response of H. medicinalis to this growth factor is explained by the presence of two specific VEGF-like receptors (Flt-1/VEGFR-1 and Flk-1/VEGFR-2) as demonstrated by immunohistochemistry and biochemical analysis. The VEGF-like produced by this annelid following surgical stimulation determines not only blood vessel formation, proliferation of vascular endothelial cells but also an increase of cytoplasmic calcium levels. The administration of specific VEGF receptor antibodies can inhibit angiogenesis in leeches previously stimulated with VEGF.

From the basic expression vector p/hVEGF165, containing a cDNA sequence encoding the 165-amino-acid isoform of human vascular endothelial growth factor (VEGF165), we generated an improved construct (p163/hVEGF165) by subcloning at the 5' exact end of the VEGF165 cDNA a 163-bp IRES element belonging to the 1,014-bp, 5'-untranslated region of the murine VEGF gene. This IRES structure caused enhanced synthesis and increased secretion of the mature protein both in HEK-293 and in COS-7 cells, when compared to the basic construct. Both p/hVEGF165 and p163/hVEGF165 vectors were tested for in vivo angiogenic activity on a novel hirudinean model. As expected, the p/hVEGF165 vector injected as naked DNA was able to induce angiogenesis in the non-vascularized muscular tissue of Hirudo medicinalis. This model also allowed us to monitor intracellular synthesis of VEGF165 and subsequent interstitial secretion from muscle cells. Interestingly, significantly larger muscle tissue areas underwent marked angiogenesis when the improved vector p163/hVEGF165 was injected in H. medicinalis. It thus appears that the p163/hVEGF165 construct allows enhanced expression of the human VEGF165 gene, which in turn is responsible for increased secretion of biologically active growth factor by transduced cells. Since a naked-DNA vector very similar to p/hVEGF165 was recently found to be very active in humans for treatment of heart and limb ischemia, we suggest that our improved construct might be further tested in view of potential therapeutic applications.

The innate immune response is the first line of defence strategies in invertebrates against attack of infectious agents. A detailed analysis of the immune mechanisms involved in annelids has been performed in oligochaets, but few data are available in polichaets and hirudineans. The aim of this review is to describe the responses of leeches to different kinds of stimuli (infections following non-self agent attacks, surgical lesions, grafts). Furthermore, the use of this invertebrate as a novel experimental model to be used to screen drugs and genes, which are responsible for positive and negative modulation of angiogenesis, is discussed.